Formins are multidomain proteins that participate in a wide range of cytoskeletal processes. They are required for cell polarity, cell migration, cytokinesis, and morphogenesis in all eukaryotes. The defining feature of formin proteins is the Formin Homology 2 (FH2) domain, which directly nucleates actin filaments and remains processively associated with the barbed end of the filament as it grows. The actin assembly activity of formins must be tightly regulated. In the Diaphanous-related formins (DRFs), binding of Rho-family GTPases is one mechanism that affects release of autoinhibitory interactions to activate the FH2 domain. Because they reorganize the actin cytoskeleton in response to diverse cellular signals, formins are of central importance in cell biology and to human health. Defects in formin proteins result in failed cytokinesis and abnormal development. Our long-term goal is to understand at a structural level the regulated assembly of actin filaments by formin proteins and their binding partners. In the previous project period, we determined crystal structures representing most of the known functional domains of diaphanous-related formins, including the N-terminal regulatory region, and the FH2 and inhibitory DAD domains. In this renewal, we build on this foundation to elucidate the structure of the autoinhibited state and to further dissect the structural requirements for nucleation and processive capping. To better understand how formins work in specific cellular contexts, we expand our studies to include two genetically and biochemically well-validated formin partner proteins, the yeast protein Bud6 and the metazoan protein Spire. Bud6 is a key binding partner and regulator of the formin Bni1 in yeast, and Spire is an actin nucleator that cooperates with the formin Cappuccino in the fly or formins Fmn-1 and Fmn-2 in mammals. These studies will reveal both distinct, formin-specific regulatory mechanisms and common principles that are applicable to many formin proteins. PUBLIC HEALTH RELEVANCE: Formin proteins are critical components of the apparatus that cells use to reorganize their internal structure, or cytoskeleton. Particular formin proteins carry out such reorganization for particular needs, such as cell division or to allow movement of the cell. Genetic defects in formins can lead to deafness, infertility and developmental abnormalities. Blocking the action of formins might be a useful approach to treating metastatic cancers. We are working to understand the structure and regulation of formins at a basic level; in the long term our studies will contribute to a better understanding of formin function in these and other diseases.